Analyst pc densitometric software

HaCaT cells (1 × 10⁵ cells/well in 6-well plates) were cultured in DMEM with or without compound 4 (0–2.5 µM) for 12 and 24 h. The cells were lysed with RIPA buffer containing 1% protease inhibitor and 1% phosphatase inhibitor on ice for 45 min. Then, the total protein from each sample was determined by bicinchoninic acid (BCA) protein assay kit (Cat No. 23225, Thermo Fisher Scientific, Rockford, IL, USA). The equal amount (40 µg) of protein sample was mixed with loading buffer and heated at 95 °C for 5 min before loaded to 10% sodium dodecyl sulfate–polyacrylamide gels (SDS-PAGE). The proteins were transferred onto PVDF membrane, which was further blocked with 5% BSA in TBST (25 mmol/L Tris–HCl; pH 7.4, 125 mmol/L NaCl, 0.1% Tween 20) at room temperature for 1 h. The primary antibody specific to SRD5A1 (1TFS-AB-PA575919, Thermo Fisher Scientific, Rockford, IL, USA) in dilution 1:1000 was added onto the membrane and incubated overnight at 4 °C. Then the membrane was washed 3 times × 7 min with TBST following with the immersion in the specific secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. The signal of target protein was determined by using chemiluminescence HRP substrate (Thermo Scientific, Rockford, IL, USA) and the intensity of target protein was analyzed by analyst/PC densitometric software (Bio-Rad, Hercules, CA, USA).

MNT1 cells (1 × 10⁵ cells/well) in 6-well plates were cultured in complete DMEM with or without 50 μM cajanin for 24–72 h. The cells were harvested and lysed with RIPA buffer containing 1% protease inhibitor and 1% phosphatase inhibitor for 45 min on ice. Then, the equal protein samples determined by BCA assay kit were mixed with loading buffer, heated at 95 °C for 5 min and further subjected to 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). The separated proteins in polyacrylamide gels were transferred onto nitrocellulose membranes and blocked with 5% skim milk in TBST (25 mmol/L Tris-HCl; pH 7.4, 125 mmol/L NaCl, 0.1% Tween 20) at room temperature for 1 h. The membranes were incubated overnight with specific primary antibody at 4 °C then washed 3 times × 7 min with TBST. Horseradish peroxidase (HRP)-conjugated secondary antibody was added onto the membrane to interact with respective primary antibody for 2 h at room temperature. The signal from the target protein was visualized by using an enhanced chemiluminescence HRP substrate (Thermo Scientific, Rockford, IL, USA). The intensity of protein signal was quantified using analyst/PC densitometric software (Bio-Rad, Hercules, CA, USA).